In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

The overall goal of this procedure is the in vitro synthesis of modified mRNA for induction of protein expression in cells. This is accomplished by first amplifying the plasmid insert or the coding DNA sequence and adding a poly T tail of 120 thymidine using polymerase chain reaction. The second step is the in vitro transcription of the generated DNA to mRNA with poly adenine tails.

Next, the template DNA is removed by D'S treatment of the in vitro transcription reaction mixture. The final step is the Antarctic phosphatase treatment of the produced mRNA to remove five prime phosphates. Ultimately, cells are transfected by the generated mRNA, lipid complexes.

Fluorescent microscopy and flow cytometry analysis are used to show the synthesis of enhanced GFP in cells. The main advantage of this technique over existing methods like viral transfect, is the lack of integration into the host cell genome, which prevents the risk of oncogenesis. This method can help to produce missing or defective proteins in cells and can be used for vaccination treatment of genetic disorders.

And in the field of regenerative medicine, Demonstrating the procedure will be steinle a grad student from my laboratory. Prior to the start of this procedure, the plasmids containing the required coding DNA sequences or CDS were amplified in e coli and isolated as described in the protocol text. In this example, the CDS of interest is enhanced green fluorescent protein or EGFP to simultaneously amplify and add a poly detail to the CDS of EGFP.

Prepare a PCR mixture as detailed in the protocol text. Place the PCR tube in a thermocycler and run the PCR cycling protocol that is described in the protocol text. When the PCR reaction is complete, clean up the PCR reaction using a commercially available PCR purification kit and dilute the DNA using 20 microliters of nuclease free water.

To check the quality of the PCR product, perform DNA gel electrophoresis and visualize the DNA bands on an imaging system. A clear DNA band with a length of approximately 1, 100 base pairs should be detected during in vitro transcription or IVT. The DNA generated earlier will be transcribed to mRNA.

Prepare the NTP cap analog mixture as shown here. Mix the NTP cap analog mixture thoroughly by vortexing. Then spin it down Briefly assemble the IVT reaction mixture as described in this table.

Mix the IVT reaction mixture thoroughly by gently pipetting up and down. Then centrifuge the PCR tube briefly to collect the mixture at the bottom of the tube. Incubate the IVT reaction mixture at 37 degrees Celsius for three hours in a thermo mixer.

After three hours, remove the template DNA by adding one microliter of DNAs to the IVT reaction mixture. Mixing it well and incubating at 37 degrees Celsius for 15 minutes. Purify the reaction mixture using an RNA purification kit and dilute the modified MR NA from the spin column membrane twice with 40 microliters of nuclease free water, the modified mRNA generated by IVT must be treated with phosphatase to remove five prime tri phosphates, which can lead to immune activation.

To begin this procedure, add nine microliters of 10 x Antarctic phosphatase reaction buffer to 79 microliters of a purified mRNA solution. Subsequently, add two microliters of Antarctic phosphatase and gently mix the sample. Incubate the mixture at 37 degrees Celsius for 30 minutes.

After 30 minutes, purify the reaction mixture using an RNA purification kit and dilute the modified mRNA from the spin column membrane twice with 50 microliters of nuclease free water. After measuring the concentration of the modified mRNA, adjust the concentration to 100 nanograms per microliter by adding nuclease free water. Check the quality of the synthesized modified mRNA by RNA gel electrophoresis and visualize the ladder and mRNA bans on a UV trans illuminator.

A clear mRNA band with a length of approximately 1, 100 base pairs should be detected. Prepare the HEC 2 9 3 cells for transfection with the synthesized modified mRNA by plating two times. Center the five cells per well of a 12 Well plate incubate the cells overnight at 37 degrees Celsius in a cell incubator On the following day, the cell should have reached 80 to 90%confluence.

Generate the lipo plexus for transfection. Prepare enough transfection mixture for all wells to be transfected each well of a 12 well plate requires 2.5 micrograms of modified mRNA, two microliters of lipid transfection reagent and 473 microliters of opt one. Reduce serum medium.

Mix the components gently by pipetting as a negative control. Prepare a transfection mixture without Mr.NA, incubate the transfection mixtures at room temperature for 20 minutes to generate lipo plexes. For transfection.

Wash the cells with 500 microliters of DPBS per. Well add 500 microliters of transfection mixture to each well of the 12 well plate incubate the cells for four hours at 37 degrees Celsius and 5%carbon dioxide after four hours, aspirate the transfection mixture and add one milliliter of complete cell culture. Medium to the cells in each well.

Incubate the cells for 24 hours in the cell incubator. Subsequently perform fluorescence microscopic analysis of the cells using a fluorescence microscope to prepare cells for flow cytometry. Remove cell culture medium from the wells and wash each well with one of DPBS without calcium and magnesium.

Add 500 microliters of TRIPSIN EDTA per well to detach the cells from the surface of the cell culture plates. Subsequently, add 500 microliters of trypsin neutralizing solution per well to inactivate tryin centrifuge the detached cells for five minutes at 300 times G room temperature. After removing the supinate Reese, suspend the cell palate in 150 microliters of cell fixation solution and transfer the cell suspension into a flow cytometry tube.

Analyze the percentage of EGFP expressing cells and the fluorescence intensity using the geometric mean of fluorescence intensity. Protein expression over time is also assessed by flow cytometry analysis of the EGFP expressing cells at one, two, and three days after transfection as described in the protocol text, the functionality of the generated EGFP mRNA was tested by transfection of HEC 2 9 3 cells. The production of EGFP in the cells was detected 24 hours after transfection using fluorescence microscopy.

Here, a face contrast image of the cells is shown next to a fluorescence image of the cells. EGFP expression in the cells was also detected by flow cytometry 24 hours after transfection. The pink line represents cells without mRNA, transfection, and the blue line represents EGFP positive cells.

After EGFP mRNA transfection, 93.91%of all measured cells were positive, and the geometric mean of fluorescence intensity was 720.16. The duration of protein expression in the HC 2 93 cells was evaluated by measuring EGFP expression one, two, and three days after mRNA. Transfection protein expression was highest in the cells 24 hours after transfection.

The amount was reduced by 1.6 fold each following day, but even after three days, the cells contained EGFP Once mastered. This technique can be done in two days if it is performed properly. After watching this video, you should have a good understanding of how to produce stabilized modified MRNA for the induction of desired protein expression in cells.